Novel five nucleotide deletion in dysferlin leads to autosomal recessive limb‐girdle muscular dystrophy

Abstract Muscular dystrophy (MD) is a genetic disorder that causes progressive muscle weakness and degeneration. Limb‐girdle muscular dystrophy (LGMD) is a type of MD that mainly causes muscle atrophy within the shoulder and pelvic girdles. LGMD is classified into autosomal dominant (LGMD‐D) and autosomal recessive (LGMD‐R) inheritance patterns. Mutations in the Dysferlin gene (DYSF) are common causes of LGMD‐R. However, genetic screening of DYSF mutations is rare in Taiwan. Herein, we identified a novel c.2867_2871del ACCAG deletion and a previously reported c.937+1G>A mutation in DYSF from a Taiwanese family with LGMD. The primary symptoms of both siblings were difficulty climbing stairs, walking on the toes, and gradually worsening weakness in the proximal muscles and increased creatine kinase level. Through pedigree analysis and sequencing, two siblings from this family were found to have compound heterozygous DYSF mutations (c. 937+1G>A and c. 2867_2871del ACCAG) within the separated alleles. These mutations induced early stop codons; if translated, truncated DYSF proteins will be expressed. Or, the mRNA products of these two mutations will merit the nonsense‐mediated decay, might result in no dysferlin protein expressed. To our knowledge, this is the first report of a novel c.2867_2871del ACCAG deletion in DYSF. Further research is required to examine the effects of the novel DYSF mutation in Taiwanese patients with LGMD.

Muscular dystrophy (MD) is an inherited genetic disorder with muscle weakness and degeneration (Mercuri et al., 2019).Limb-girdle muscular dystrophy (LGMD) is a type of MD characterized by progressive voluntary muscle atrophy and hip and shoulder faintness, and may affect other body muscles.
Recently, the Universal Mutation Database for Dysferlin, a locus-specific database developed with the UMD® software, has been compiled to provide up-to-date information about mutations of the DYSF gene (Blandin et al., 2012).It aims to provide accessible information and easy ways for researchers to report their findings.Numerous DYSF mutations have recently been identified in LGMD and classified as LGMD-R2 (dysferlinopathy) (Millay et al., 2009;Saito et al., 2002;Santos et al., 2010).For example, in Japan, the c.2997G>T mutation in DYSF was associated with late-onset, proximal dominant forms of dysferlinopathy (Takahashi et al., 2013).Recently, more comprehensive genetic screening, and the diagnostic methods of MD have improved in Taiwan (Liang et al., 2020;Lin et al., 2021).However, DYSF mutationrelated LGMD data are still rare in Taiwan, resulting in a lack of practical clinical approaches for diagnosing LGMD.
In this study, we identified a novel c.2867_2871del ACCAG deletion in DYSF in two siblings from a family with LGMD, which was combined with a c.937+1G>A mutation in the other allele.Family inheritance patterns were revealed, and the c.2867_2871del ACCAG deletion and c.937+1G>A mutation in DYSF induced early stop codons which may generate the truncated DYSF proteins, if translated.Or, the mRNA products of these two mutations could cause the nonsense mediated decay, resulting in no dysferlin expressed.To our knowledge, this is the first report of a novel c.2867_2871del ACCAG deletion in DYSF.

| Patient description
The study had been reviewed and approved by the Research Ethics Review Committee of the Cardinal Tien Hospital for all bio-clinical specimens (CTH-3-5-0332021/10/30).There are six members in the LGMD family (refer to Figure 1) were enrolled in this study and the written informed consent was obtained from all patients.Numbered 4 patient were the onset of the disease at the age of 14, gradually, and is 41 years old, now.The primary symptoms were experience of difficulties climbing stairs, walk on the toe and gradually worsening weakness in the proximal muscles, particularly in the calf and thigh.The serum lactate dehydrogenase (LDH) and creatine kinase (CK) were evaluated at diagnosis.Besides, electromyography, cardiology and respiratory tests have analyzed.
2.2 | DNA preparation, whole-exon sequence, and data analysis Blood samples were collected from the subjects (Figure 1), and DNA was isolated using a blood DNA extraction kit (Cat.51104) (Qiagen, the Netherlands), following the manufacturer's standard protocol.The DNA samples from the proband underwent whole-exon sequencing, whereby the genomic DNA was enriched using an Agilent SureSelect Human All Exon Enrichment Kit V6 array (Cat.5190) (Agilent Technologies, Santa Clara, CA, USA), and the fragmented DNA was enhanced and subjected to sequencing using an Illumina NextSeq550 system (Illumina, San Diego, CA, USA).The sequencing depth for each participant was >100fold.The NextGENe software was used for alignment, variant calling, and filtering (SOFTGENETICS, State College, PA, USA).

| Bioinformation evaluation and three-dimensional (3D) structure prediction
To predict the translated amino acid sequence or molecular weight of DYSF with c.937+1G>A or c.2867_2871del ACCAG mutations, the ExPASy Translate server (http:// web.expasy.org/ trans late/ ) together with ExPASy Compute pI/Mw (https:// web.expasy.org/ compu te_ pi/ ) was used according to the manufacturer's instructions.An artificial intelligence system, AlphaFold, developed by DeepMind, was used to predict the 3D structure of mutated DYSF amino acid sequences (Spadafora et al., 2022).The 3D structural models of DYSF were coded into the wild-type file O75923 model.

| RESULTS
3.1 | Identified of a novel c.2867_2871del

ACCAG deletion in DYSF in LGMD patients
First, the proband (Number 4; Figure 1), currently 41 years old, experienced disease onset at the age of 14 years.The primary symptoms were difficulty climbing stairs, walking on the toes, and gradually worsening weakness of the proximal muscles, particularly the calf and thigh.Serum lactate dehydrogenase and creatine kinase of the patient were elevated as 325 and 2484 U/L, respectively.And, no any abnormalities were found within the cardiological and respiratory tests.Following, the patient was diagnosed with muscular dystrophy.To explore the possible genetic mutation in the patient (number 4), whole exons sequencing was applied.DYSF mutations, c.937+1G>A (exon 10) and c.2867_2871del ACCAG (exon 27), were identified.To track the inheritance of this family, the exons 10 and 27 of DYSF were sequenced in the other family members (Figure 1).The inheritance patterns of the two DYSF mutations in the family are shown in Figure 1 (right panel).Among the siblings, both types of mutations (c.937+1G>A and c.2867_2871del ACCAG) were present in siblings 3 and 4 (current patient).The younger brother was a carrier of a single c.937+1G>A mutation (number 6), and the younger sister did not exhibit either of the two mutation phenotypes (number 5).The father and mother carried the c.937+1G>A and c.2867_2871del ACCAG mutations, respectively.
Herein, we identified a novel deletion, c.2867_2871del ACCAG, and a previously described c.937+1G>A mutation in DYSF in a family member with LGMD.

| The c.937+1G>A mutation in DYSF activate a cryptic splice donor site
In a previous study, the c.937+1G>A mutation in DYSF was suggested to be pathogenic through alternative splicing.To evaluate the hypothesis, the RNA transcripts were isolated from blood and subjected to RT-PCR analysis in Figure 2a (left panel).The c.937+1G>A disrupts the splicing donor site between exon 9 and exon 10 and induces a cryptic splicing donor site in intron 9 in Figure 2a (right panel).Abnormal splicing results in the abnormal transcripts (the size of PCR product: 1078 bp), which inserts 580 bp from intron 9 in patients with the c.937+1G>A mutation, compared to the 498 bp DNA fragment in control.The novel transcript therefore encodes a truncated protein lacking the large part of c-terminal DYSF.Regarding the new c.2867_2871del ACCAG deletion, RT-PCR is currently less sensitive in detecting a 5-nucleotide deletion, and mice with the c.2867_2871del ACCAG deletion are in the process of being designed.To determine whether DYSF mutations affect its protein if translation, ExPASy prediction model was applied.The expression of wild type DYSF gene resulted in a 237 kDa protein (Figure 2b left panel).Translation prediction revealed, that the RNA transcript resulting from abnormal splicing, was translated into a small truncated DYSF protein with predicted M.W. 38 kDa (Figure 2b upper right panel).By contrast, c.2867_2871del ACCAG is a novel deletion that has not been reported in the literature, international dysferlin database, or other genetic disease databases.Base on the deleted five nucleotides, a frameshift was logically assumed to induce an early stop codon during translation.An early stop codon was presumed using translation prediction software, resulting in a truncated DYSF protein (predicted M.W. 108 kDa) (Figure 2b  in spermatids of Caenorhabditis elegans (Achanzar & Ward, 1997).In normal muscles, DYSF proteins are enriched in membrane patches and respond to sarcolemma injury (Covian-Nares et al., 2010).When membrane disruption occurs, a rapid Ca 2+ flood triggers the aggregation of DYSF-carrying repair vesicles and transports them toward the disruption site to reseal the membrane (Azakir et al., 2010).By contrast, abnormal accumulation of vesicles within sub-sarcolemmal region is in the Dysferlin-null muscle, due to lack of precise vesicle regulation (Bansal et al., 2003).A recent study also showed that DYSF can stabilize stress-induced Ca 2+ signaling in the transverse tubule membrane (Han & Campbell, 2007) and regulate Ca 2+ homeostasis in the skeletal muscle (Kerr et al., 2014).In this study, we identified c.2867_2871del ACCAG deletion and c.937+1G>A mutation of DYSF, which were existed in separated alleles in the patient.The c.937+1G>A mutation in DYSF is predicted to generate a premature stop codon that could result in nonsense-mediated decay of the transcript with no dysferlin protein expressed.If translated, the shorter open reading frame is predicted to generate a truncated form (38 kDa) of dysferlin.Another novel c.2867_2871del ACCAG deletion of DYSF may create a frameshift in the transcript, which possible also merit the process of nonsense-mediated decay, and resulting in no dysferlin expressed.Alternatively, this deletion might also result in the expression of a truncated 108 kDa protein if translated.Briefly, the mRNA products of these two mutations, c.937+1G>A or c.2867_2871del ACCAG, might translate and generate the truncated dysferlin protein with 38 kDa or 108 kDa separately if the nonsense-mediated mRNA decay is not operative.Compared to the wild-type dysferlin protein (237 kDa), only the N-terminal portion of the truncated dysferlin protein might be expressed.In addition, the mRNA expression of the c.937+1G>A mutation seems to possibly be higher than that of the c.2867_2871del ACCAG deletion.However, the precise mRNA contents or even protein abundances of the c.937+1G>A mutation and c.2867_2871del ACCAG deletion needs to be evaluated in the future.Mice with c.2867_2871del ACCAG deletion or mice with two alleles carrying either the c.937+1G>A or the c.2867_2871del ACCAG mutation are in the processes.The precise molecular defects form underlying c.937+1G>A and c.2867_2871del ACCAG mutations remain unidentified and will be evaluated in the future.
Past decade, αtubulin was identified as a novel DYSF binding partner using affinity purification followed by liquid chromatography/mass spectrometry (Azakir et al., 2010;Demonbreun et al., 2014).The cellular function of microtubules, composed of α and β tubulin, can be regulated by posttranslational modifications.A recent study showed that DYSF interacts with histone deacetylase 6 through the C2D domain, preventing αtubulin deacetylation in cos-7 cells and myoblasts.In addition, increased levels of αtubulin acetylation in DYSF-expressing cells and myoblasts may enhance myotube formation (Balasubramanian et al., 2014).How mutations c.937+1G>A and c.2867_2871del ACCAG damage the construction of myotube during myogenesis remains to be evaluated.
Furthermore, RT-PCR and Northern blot analyses revealed that DYSF mRNA was also expressed in endothelial cells such as the bovine aorta and human umbilical vein endothelial cells.Knockdown of dysferlin using siRNA in subconfluent endothelial cells caused deficient adhesion, suggesting the possibility of DYSF in angiogenesis (Sharma et al., 2010).A recent study found that the loss of DYSF in human monocytes strongly reduced adhesion and increased motility, which might influence the infiltration of dysferlinopathy muscles (de Morrée et al., 2013).Hence, DYSF may modulate the adhesion of inflammatory cells and even angiogenesis during muscle repair.Whether the c.937+1G>A or c.2867_2871del ACCAG mutations facilitate the infiltration and accumulation of inflammatory cells and delay tissue repair in myotubes remains to be clarified.

| CONCLUSIONS
Many patients harboring DYSF gene mutations have been reported.In this study, we focus on identification of DYSF mutations in Taiwanese cases with LGMD.Specifically, we identified a pathogenic c.2867_2871del ACCAG of DYSF mutation that first reported here.Further understanding the molecular effects of DYSF mutation sites will be addressed in the focus on modulating inflammation together with skeletal muscle repair, following this study.

AUTHOR CONTRIBUTIONS
Yen-Lin Chen, Yi-No Wu, Ying-Hung Lin, and Wen-Bin Wu conceived and designed the study; Yen-Lin Chen performed experiments and analyzed the data; Yen-Lin Chen, Wen-Bin Wu, Pei Wang, Ping-Keung Yip, Yi-No Wu, Ying-Hung Lin, and Wen-Bin Wu interpreted the experimental results; Yen-Lin Chen Yi-No Wu, Ying-Hung Lin, and Wen-Bin Wu prepared the figures; Yen-Lin Chen and Wen-Bin Wu drafted the manuscript; Yi-No Wu and Ying-Hung Lin edited and revised the manuscript.
lower right panel). 4 | DISCUSSION DYSF shares sequence homology with fer-1, which mediates vesicle fusion with the plasma membrane F I G U R E 2 Effects of DYSF c.937 + 1G > A mutation on the splicing process.(a) Graphical presentation of the wild-type and aberrantly spliced transcripts using RT-PCR from normal (N) and DYSF c.937 + 1G > A patient (P; number 6) left panel).The DYSF c.937 + 1G > A mutation induced alternative splicing in vivo (right panel).RT-PCR fragments containing exons 10-14 (498 bp), and alternative processes induced alternative splicing (1078 bp), which resulted in the insertion of 580 bp (partial intron 10), shown as a gray box.(b) Prediction of mutant and full length DYSF translation.The translated amino acid of DYSF with wild type (left), c.937 + 1G > A (upper right), and c.2867_2871del ACCAG (lower right) mutation sequences were obtained by the ExPASy Translate server (https:// web.expasy.org/ trans late/ ) and the potent protein models were predicted by the WEISS-MODEL with the wild type template of dysferlin (O75923).